Genetic markers for bone mass

ABSTRACT

The invention provides an agent for preventing or treating arthritis, a cartilage protecting agent, a joint destruction inhibitor and a synovial membrane growth inhibitor comprising an anti-FGF-8 neutralizing antibody as an active ingredient, as well as a diagnostic agent of arthritis comprising an anti-FGF-8 antibody as an active ingredient and a method for judging arthritis using the antibody.

The present invention relates to methods for genetic analysis of bonemineral density and susceptibility to disorders which are related tobone mass. It further relates to materials for use in such methods.

BACKGROUND ART

Genetic factors play an important role in the pathogenesis ofosteoporosis—a common disease characterised by reduced bone mass,microarchitectural deterioration of bone tissue and increasedsusceptibility to fragility fractures ¹. Bone mineral density (BMD) isan important predictor of osteoporotic fracture risk and evidence fromtwin and family studies suggests that between 50%-85% of the variance inBMD is genetically determined ²⁻⁴. However the genes responsible forthese effects are incompletely defined. BMD is a complex trait, which islikely to be regulated by an interaction between environmental factorssuch as diet and exercise several different genes, each with modesteffects on BMD.

A wide variety of candidate genes have been studied so far in relationto BMD, including the vitamin D receptor ⁵, the estrogen receptor ⁶, andthe COLIA1 gene ⁷. Current evidence suggests that allelic variation inthese genes accounts for only a small portion of the variance in BMDhowever B indicating that most of the genes which regulate BMD remain tobe discovered.

Linkage studies in humans have mapped three Mendelian traits that areassociated with abnormalities of BMD to a region of chromosome 11q12-13.These are osteoporosis-pseudoglioma syndrome⁹; autosomal recessiveosteopetrosis¹⁰ and high bone mass¹¹. This region of chromosome 11 wasalso found to be linked to BMD in normal female sibling pairs¹²,indicating that allelic variation of genes within this region may play arole in regulating BMD.

Recent work has also shown evidence of linkage between a polymorphism atthe TCIRG1 locus and femoral neck BMD in healthy premenopausal sib-pairs(Carn et al (2002) J Clin.Endocrinol.Metab 87:3819-3824).

However, the results from such linkage analysis are only able tolocalise the phenotypic effects to within regions of millions of basepairs and do not identify the gene or genes responsible for thephenotypic effect observed. Thus no clear association, as distinct fromlinkage, has previously been demonstrated between markers in this regionand regulation of BMD in a normal population. The genotyping of suchgenetic markers would be useful as markers of bone mass and hence, forexample, susceptibility to osteoporotic fractures.

DISCLOSURE OF THE INVENTION

The present inventors have demonstrated that allelic variation in theTCIRG1 gene in 11q12-13 contributes to regulation of bone mass in normalindividuals.

The TCIRG1 gene is known to encode a 116Kd subunit of the osteoclastspecific vacuolar proton pump. It is a component of the vacuolar-ATPasecomplex expressed in the osteoclast ruffled border and is responsiblefor transport of H+ ions into the resorption lacuna, where the low pHplays a role in dissolving hydroxyapatite crystals¹⁷. TCIRG1 mutationshave previously been shown to be present in approximately 60% ofindividuals with infantile osteopetrosis^(13;14). However they were notknow to be associated with regulation of bone mass in normalindividuals.

Briefly, the present inventors studied the relationship between bonemineral density (BMD) and TCIRG1 polymorphisms in a population basedcohort of several hundred perimenopausal Scottish women. They identifiedfive novel polymorphisms at the TCIRG1 locus; two in the promoter; onein exon 4, one in intron 4 and one in intron 11.

The inventors demonstrated a significant association between the G9326Agenotype and BMD at the lumbar spine (p=0.01) and femoral neck (p=0.03).G9326A is within the promoter, within a consensus recognition site forthe AP1 transcription factor.

The association remained significant after correcting for age, weight,height, menopausal status/HRT use and smoking (p=0.008 for spine BMD andp=0.03 for hip BMD) and homozygotes for the “G” allele had BMD valuessignificantly higher than individuals who carried the “A” allele at bothspine (p=0.007) and hip (p=0.047). Subgroup analysis showed that theassociation between G9326A and BMD was restricted to premenopausalwomen, who comprised 50.6% of the study group.

The five polymorphisms showed strong and highly significant linkagedisequilibrium with each other in the population, with the exception ofC14242T where linkage disequilibrium was only observed with A14286G.

Thus it appears that common allelic variants (allele frequency >0.05) ofthe TCIRG1 gene can account for at least part of the heritable componentof BMD, possibly by affecting peak bone mass. The TCIRG1 polymorphismsare thus useful as genetic markers e.g. for identifying people with lowBMD, so that these individuals could be targeted for treatment toprevent osteoporosis.

BRIEF DESCRIPTION OF THE INVENTION

At its most general, the present invention provides methods forassessing bone mass (e.g. peak bone mass) and particularly BMD (e.g.lumbar spine BMD or femoral neck BMD) in an individual, the methodscomprising using a TCIRG1 marker, particularly a polymorphic marker toassess this trait.

In preferred embodiments these methods may be used to assess thesusceptibility of the individual to disorders which are to some extent(wholly or partly) related BMD. Such disorders are hereinafter termed“BMD-related disorders” and the methods and materials herein may also beused for the diagnosis andor prognosis for them.

Preferably, the present invention is concerned with disorders associatedwith low BMD, especially osteoporosis and related disorders. Forexample, the methods of the present invention may be used to determinethe risk of certain consequences of relatively low BMD, such as todetermine the risk of osteoporotic fracture (McGuigan et al (2001)Osteoporosis International, 12, 91-96).

The method may comprise:

-   -   (i) providing a sample of nucleic acid, preferably genomic DNA,        from an individual, and    -   (ii) establishing the presence or identity of one or more TCIRG1        (polymorphic) markers in the nucleic acid sample,    -   plus one or more further steps to calculate a risk of        osteoporotic fracture in the individual based on the result of        (ii).        Predicting Risk of Osteoporotic Fractures

The methods of the present invention may be used to attribute a likelyBMD value to the individual based on the result established at (ii).

Alternatively or additionally they may be used in prognostic tests toestablish, or assist in establishing, a risk of (developing an)osteoporotic fracture, which is the major clinical expression ofosteoporosis. Methods for making such predictions are well known tothose skilled in the art and the present disclosure may be used inconjunction with existing methods in order to improve their predictivepower. Other known predictors include BMD, weight, age, sex, clinicalhistory, menopausal status, HRT use, various SNPs and so on. Thediagnosis of osteoporosis (and prognosis of fracture) is reviewed byKanis et al (1994) J Bone and Mineral Res 9,8: 1137-1141.

McGuigan et al (2001) supra disclose predictive methods based on acombination of bone densitometry and genotyping (in that case COLIA1genotyping). Individuals were classified as either high or low risk onthe basis of these two methods, which were inter-related butindependently predicted risk of sustaining osteoporotic fractures. Thus,by analogy, the present TCIRG1 test may be predictive independently ofBMD scores.

Marshall (1996) BMJ 312: 1254-1259 discloses a meta-analysis of how BMDmeasures predict osteoporotic fractures and attributed relative riskvalues and confidence intervals to various BMD measurements. The paperrefers to a number of other risk factors for fracture. Cummings et al(1995) N Engl J Med 332: 767-73, also reviews risk factors (in that casefor hip fracture in white woman)

All of these papers, inasmuch as they may be utilised by those skilledin the art in practising the present invention, are hereby incorporatedby reference.

Thus preferred aspects of the invention will involve establishing orutilising one or more further measures which are predictive ofosteoporotic fracture and defining a risk value (e.g. low, medium, high)or relative risk values or odds ratios (adjusted, for instance, againstthe population of that age and optionally sex) and optionally aconfidence value or interval, based on the combination of these.Statistical methods for use in such predictions (e.g. Chi-square test,logistic regression analysis and so on) are well known to those skilledin the art. In a preferred embodiments a battery of tests (bothgenotyping and phenotyping) will be employed to maximise predictivepower.

The methods may further include the step of providing advice toindividuals characterised as being above low or medium risk, in order toreduce that risk (e.g. in terms of lifestyle, diet, and so on).

Particular methods of detecting polymorphisms in nucleic acid samplesare described in more detail hereinafter.

Nucleic Acid Sample

The sample from the individual may be prepared from any convenientsample, for example from blood or skin tissue. The DNA sample analysedmay be all or part of the sample being obtained. Methods of the presentinvention may therefore include obtaining a sample of nucleic acidobtained from an individual. Alternatively, the assessment of the TCIRG1polymorphic marker may be performed or based on an historical DNAsample, or information already obtained therefrom e.g. by assessing theTCIRG1 polymorphic marker in DNA sequences which are stored on adatabank.

Where the polymorphism is not intronic the assessment may be performedusing mRNA (or cDNA), rather than genomic DNA.

Choice of Individual

Where the present invention relates to the analysis of nucleic acid ofan individual, such an individual may be entirely symptomless, or may beone who has a BMD-related disorder, or is considered to be at risk fromBMD-related disorder such as osteoporosis (e.g. by virtue of otherdeterminants e.g. age, weight, menopausal status, HRT use etc. Asdescribed in the results below, although the association with preferredmarkers was demonstrated in the whole population, subgroup analysisrevealed that the effect was primarily driven by an association in thepremenopausal population. This would be consistent with a model wherebythe TCIRG1 allele affects peak bone mass rather than postmenopausal boneloss,

The method may be used to assess risk within a population by screeningindividual members of that population.

Preferred Markers

It is preferred that the polymorphic marker is a single nucleotidepolymorphism (SNP), which may be in an intron, exon or promoter sequenceof the TCIRG1 gene. Preferably it will be a common allelic variant(allele frequency >0.05).

Preferred polymorphisms are as follows:

-   -   G9326A: situated in the promoter.    -   G9508A: situated in the promoter.    -   C14242T: situated in exon 4.    -   A14286G: situated within intron 4    -   G19031A: situated within intron 11.

It should be noted that all polymorphisms are, for convenience, numberedin relation to the latest sequence accession at the time of filing(LOCUS AP002807, 63433 bp DNA Linear PRI 24-JAN-2002, DEFINITION Homosapiens genomic DNA, chromosome 11q clone:RP11-802E16, completesequences—revised Jul. 5, 2002). Using an earlier accession (AF033033)14242, 14286 & 190031 were at gene positions 3856, 3900 & 8645respectively.

Annex I shows sequence of the TCIRG1 gene (as taken from a BAC clone).The promoter SNPs 9326 and 9508 are at positions 2648 and 2830respectively. Based on the disclosure herein the skilled person is wellable to identify the position of the polymorphisms of the invention inthe TCIRG1 sequence.

Thus preferred SNPs for analysis are at any one or more of the followingTCIRG1 gene positions: 9326, 9508, 14242, 14286, 19031.

More preferred are SNPs at position: 9326. The association between BMDand allelic variation at the G9326A site was highly significant at thespine (p=0.007) and at the femoral neck (p=0.03), after correcting forpotential confounding factors including age, height, weight, menopausalstatus/HRT use and smoking.

Accordingly, in one embodiment the method of the present inventioncomprises assessing in a genomic DNA sample obtained from an individualone or more TCIRG1 SNPs selected from the SNP at position 9326, or apolymorphism in linkage disequilibrium with said SNP.

In a further embodiment the method may comprise assessing two, three,four or five of the TCIRG1 SNPs. Any suitable combination of one or moremarkers may be used to assess the BMD trait.

The method of the invention may comprise, in addition to assessing oneor more TCIRG1 SNPs, or one or more polymorphisms in linkagedisequilibrium with a TCIRG1 SNP, the assessment of other polymorphismswhich are linked or associated with a BMD-related disorder.

Examples of such other polymorphisms include polymorphisms in the VDRgene and the COLIA1 gene (Uitterlinden, et al. (2001) Journal of Boneand Mineral Research).

Identity of Alleles

The assessment of an SNP will generally involve determining the identityof a nucleotide at the position of said single nucleotide polymorphism.

Preferred assessment of the SNP at position 9326 described above willestablish whether or not the individual is homozygous for the G alleleat these sites (and hence likely to have higher BMD).

For example, for SNP 9326, in relation to likely susceptibility to adisorder associated with low BMD, an individual who is A/A homozygousfor the polymorphism is classified as being at the highest risk; anindividual who is A/G heterozygous is classified as having moderaterisk; an individual who is G/G homozygous is in the lowest riskcategory.

Use of Functional Polymorphisms

Most preferred for use in the present invention are SNPs which aredirectly responsible for the BMD phenotype (“functional polymorphisms”).Intronic SNPs may, for example, be situated in regions involved in genetranscripton. SNPs may be directly responsible for the BMD phenotypebecause of an effect on the amino acid coding, or by disruption ofregulatory elements, e.g., which may regulate gene expression, or bydisruption of sequences (which may be exonic or intronic) involved inregulation of splicing, such as exonic or splicing enhancers asdiscussed below.

It is notable that of the two promoter polymorphisms, one (G9326A) issituated at a consensus recognition sequence for the transcriptionfactor AP1 (http://transfac.gbf.de/). In the presence of theG-nucleotide, the consensus AP1 site is present (TCACGGC) on the reversestrand whereas in the presence of the A nucleotide, the consensussequence is disrupted (TCATGGC).

The A14286G polymorphisms is in intron 4 of the TCIRG1 gene. Twotranscripts are derived from the TCIRG1 locus however. The osteoclastspecific form (termed ATP6i) is assembled from 20 exons, whereas anothertranscript termed TIRC7, which is more widely expressed, comprises 14exons and starts in exon 5 of the osteoclast-specific isoform. Since theA14286G polymorphism is in the proximal promoter of the shorter TCIR7transcript (intron 4 is only 82 bp long), it may influence transcriptionor splicing of TCIRG1.

A coding polymorphism in TCIRG1 has been described (at position 2827 onAF033033) which causes an arginine to tryptophan amino acid change atcodon 56 (R56W). While this polymorphism was observed in our population,it was rare (allele frequency 0.02) and therefore unlikely to explainthe effect observed.

Irrespective of these points and the precise underlying cause of theassociations described herein, those skilled in the art will appreciatethat the disclosure has great utility for genotyping of BMD inindividuals, whether through functional polymorphisms, or polymorphismswhich are in linkage disequilibrium with functional polymorphisms (whichmay be elsewhere in the TCRIG1 locus or in other genes nearby). Theinvention thus extends to the use not only of the markers describedabove, but also (for example) to polymorphic markers which are inlinkage disequilibrium with any of the markers discussed above, e.g., inlinkage disequilibrium with the preferred marker at position 9326.

Use of Other Polymorphisms

As is understood by the person skilled in the art, linkagedisequilibrium is the non-random association of alleles. Further detailsmay be found in Kruglyak (1999) Nature Genetics, Vol 22, page 139 andBoehnke (2001) Nature Genetics 25: 246-247). For example, results ofrecent studies indicate (summarised by Boehnke) that significant linkagedisequilibrium may extend for between 0.1 to 0.2 centimorgans.

The five markers described above showed strong and highly significantlinkage disequilibrium with each other in our population, with theexception of C14242T where linkage disequilibrium was only observed withA14286G.

Other polymorphic markers which are in linkage disequilibrium with anyof the polymorphic markers described above may be identified in thelight of the disclosure herein without undue burden by further analysise.g., within the TCIRG1 gene.

Thus in a related aspect, the present invention provides a method formapping further polymorphisms which are associated, or are in linkagedisequilibrium with a TCIRG1 polymorphism, as described herein. Such amethod may preferably be used to identify further polymorphismsassociated with variation in BMD. Such a method may involve sequencingof the TCIRG1 gene, or may involve sequencing regions upstream anddownstream of the TCIRG1 gene for associated polymorphisms.

In a further aspect, the present invention provides a method ofidentifying open reading frames which influence BMD. Such a method maycomprise screening a genomic sample with an oligonucleotide sequencederived from a TCIRG1 polymorphic marker as described herein andidentifying open reading frames proximal to that genetic sequence.

A region which is described as ‘proximal’ to a polymorphic marker may bewithin about 1000 kb of the marker, preferably within about 500 kb away,and more preferably within about 100 kb, more preferably within 50 kb,more preferably within 10 kb of the marker.

Materials

The invention further provides oligonucleotides for use in probing oramplification reactions, which may be fragments of the sequence shown inAnnex I, or a polymorphic variant thereof (see Tables herein).

Preferred primers are as follows:

-   -   for the promoter polymorphisms (G9326A and G9508A): Forward: 5′        ACAAGGCAGGCGCAGGACTCC and Reverse: CGGGCCTGGAAACTGAGTCAC;    -   for the exon 4 (C14242T) and intron 4 (A14286G) polymorphisms:        Forward 5′ TTGGGGCAGCAGGTGGGGCC 3′ and Reverse:        AGAGGAGAACCCCCTAGGGCTAG 3′;    -   for the intron 11 polymorphism (G19031A): Forward:        GTTCGGGGATGTGGGCCAC 3′/and Reverse: 5′ GCCCATAAGCAGGAGCAGG 3′.

Nucleic acid for use in the methods of the present invention, such as anoligonucleotide probe and/or pair of amplification primers, may beprovided in isolated form and may be part of a kit, e.g. in a suitablecontainer such as a vial in which the contents are protected from theexternal environment. The kit may include instructions for use of thenucleic acid, e.g. in PCR and/or a method for determining the presenceof nucleic acid of interest in a test sample. A kit wherein the nucleicacid is intended for use in PCR may include one or more other reagentsrequired for the reaction, such as polymerase, nucleosides, buffersolution etc. The nucleic acid may be labelled. A kit for use indetermining the presence or absence of nucleic acid of interest mayinclude one or more articles and/or reagents for performance of themethod, such as means for providing the test sample itself, e.g. a swabfor removing cells from the buccal cavity or a syringe for removing ablood sample (such components generally being sterile).

The various embodiments of the invention described above may also applyto the following: a diagnostic means for determing the risk of aBMD-related disorder (e.g. osteoporosis); a diagnostic kit comprisingsuch a diagnostic means; a method of osteoporosis therapy, which mayinclude the step of screening an individual for a genetic predispositionto osteoporosis, wherein the predisposition is correlated with a TCIRG1polymorphic marker, and if a predisposition is identified, treating thatindividual to prevent or reduce the onset of osteoporosis (such a methodmay comprise the treatment of the individual by hormone replacementtherapy); and the use, in the manufacture of means for assessing whetheran individual has a predisposition to osteoporosis, of sequences (e.g.,PCR primers) to amplify a region of the TCIRG1 gene.

Assessment of SNPs

Methods for assessment of polymorphisms are reviewed by Schafer andHawkins, (Nature Biotechnology (1998)16, 33-39, and references referredto therein) and include: allele specific oligonucleotide probing,amplification using PCR, denaturing gradient gel electrophoresis, RNasecleavage, chemical cleavage of mismatch, T4 endonuclease VII cleavage,multiphoton detection, cleavase fragment length polymorphism, E.colimismatch repair enzymes, denaturing high performance liquidchromatography, (MALDI-TOF) mass spectrometry, analysing the meltingcharacteristics for double stranded DNA fragments as described by Akeyet al (2001) Biotechniques 30; 358-367. These references, inasmuch asthey be used in the performance of the present invention by thoseskilled in the art, are specifically incorporated herein by reference.

The assessment of the polymorphism may be carried out on a DNAmicrochip, if appropriate. One example of such a microchip system mayinvolve the synthesis of microarrays of oligonucleotides on a glasssupport. Fluorescently-labelled PCR products may then be hybridised tothe oligonucleotide array and sequence specific hybridisation may bedetected by scanning confocal microscopy and analysed automatically (seeMarshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review).

Some preferred examples of such methods will now be discussed in moredetail.

Use of Nucleic Acid Probes

The method of assessment of the polymorphism may comprise determiningthe binding of an oligonucleotide probe to the nucleic acid sample. Theprobe may comprise a nucleic acid sequence which binds specifically to aparticular allele of a polymorphism and does not bind specifically toother alleles of the polymorphism. Where the nucleic acid isdouble-stranded DNA, hybridisation will generally be preceded bydenaturation to produce single-stranded DNA. A screening procedure,chosen from the many available to those skilled in the art, is used toidentify successful hybridisation events and isolated hybridised nucleicacid.

Probing may employ the standard Southern blotting technique. Forinstance DNA may be extracted from cells and digested with differentrestriction enzymes. Restriction fragments may then be separated byelectrophoresis on an agarose gel, before denaturation and transfer to anitrocellulose filter. Labelled probe may be hybridised to the DNAfragments on the filter and binding determined.

Binding of a probe to target nucleic acid (e.g. DNA) may be measuredusing any of a variety of techniques at the disposal of those skilled inthe art. For instance, probes may be radioactively, fluorescently orenzymatically labelled. Polymorphisms may be detected by contacting thesample with one or more labelled nucleic acid reagents includingrecombinant DNA molecules, cloned genes or degenerate variants thereofunder conditions favorable for the specific annealing of these reagentsto their complementary sequences within the relevant gene. Preferably,the lengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed nucleic acids areremoved from the nucleic acid:gene hybrid. The presence of nucleic acidsthat have hybridized, if any such molecules exist, is then detected.Using such a detection scheme, the nucleic acid from the cell type ortissue of interest can be immobilized, for example, to a solid supportsuch as a membrane, or a plastic surface such as that on a microtitreplate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents are easily removed.Detection of the remaining, annealed, labeled nucleic acid reagents isaccomplished using standard techniques well-known to those in the art.The gene sequences to which the nucleic acid reagents have annealed canbe compared to the annealing pattern expected from a normal genesequence in order to determine whether a gene mutation is present.

Approaches which rely on hybridisation between a probe and test nucleicacid and subsequent detection of a mismatch may be employed. Underappropriate conditions (temperature, pH etc.), an oligonucleotide probewill hybridise with a sequence which is not entirely complementary. Thedegree of base-pairing between the two molecules will be sufficient forthem to anneal despite a mis-match. Various approaches are well known inthe art for detecting the presence of a mis-match between two annealingnucleic acid molecules. For instance, RN'ase A cleaves at the site of amis-match. Cleavage can be detected by electrophoresing test nucleicacid to which the relevant probe or probe has annealed and looking forsmaller molecules (i.e. molecules with higher electrophoretic mobility)than the full length probe/test hybrid. Other approaches rely on the useof enzymes such as resolvases or endonucleases.

Thus, an oligonucleotide probe that has the sequence of a region of thenormal gene (either sense or anti-sense strand) in which polymorphismsassociated with the trait of interest are known to occur may be annealedto test nucleic acid and the presence or absence of a mis-matchdetermined. Detection of the presence of a mis-match may indicate thepresence in the test nucleic acid of a mutation associated with thetrait. On the other hand, an oligonucleotide probe that has the sequenceof a region of the gene including a mutation associated with diseaseresistance may be annealed to test nucleic acid and the presence orabsence of a mis-match determined. The presence of a mis-match mayindicate that the nucleic acid in the test sample has the normalsequence, or a different mutant or allele sequence. In either case, abattery of probes to different regions of the gene may be employed.

As discussed above, suitable probes may comprise all or part of thesequence shown in Annex I (or complement thereof), or all or part of apolymorphic form of the sequence shown in Annex I (or complement thereof(e.g., containing one or more of the polymorphisms shown in the Tables).

Those skilled in the art are well able to employ suitable conditions ofthe desired stringency for selective hybridisation, taking into accountfactors such as oligonucleotide length and base composition, temperatureand so on.

Suitable selective hybridisation conditions for oligonucleotides of 17to 30 bases include hybridization overnight at 42° C. in 6×SSC andwashing in 6×SSC at a series of increasing temperatures from 42° C. to65° C. One common formula for calculating the stringency conditionsrequired to achieve hybridization between nucleic acid molecules of aspecified sequence homology is (Sambrook et al., 1989): T_(m)=81.5°C.+16.6Log [Na+]+0.41 (% G+C)−0.63 (% formamide)−600/#bp in duplex.

Other suitable conditions and protocols are described in MolecularCloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, ColdSpring Harbor Laboratory Press and Current Protocols in MolecularBiology, Ausubel et al. eds., John Wiley & Sons, 1992.

Amplification-based Methods

The hybridisation of such a probe may be part of a PCR or otheramplification procedure. Accordingly, in one embodiment the method ofassessing the polymorphism includes the step of amplifying a portion ofthe TCIRG1 locus, which portion comprises at least one polymorphism.

The assessment of the polymorphism in the amplification product may thenbe carried out by any suitable method, e.g., as described herein. Anexample of such a method is a combination of PCR and low stringencyhybridisation with a suitable probe. Unless stated otherwise, themethods of assessing the polymorphism described herein may be performedon a genomic DNA sample, or on an amplification product thereof.

Where the method involves PCR, or other amplification procedure, anysuitable PCR primers may be used. The person skilled in the art is ableto design such primers, examples of which are shown in Table 3.

An oligonucleotide for use in nucleic acid amplification may be about 30or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specificprimers are upwards of 14 nucleotides in length, but need not be than18-20. Those skilled in the art are well versed in the design of primersfor use processes such as PCR.

Various techniques for synthesizing oligonucleotide primers are ellknown in the art, including phosphotriester and phosphodiester synthesismethods.

Suitable polymerase chain reaction (PCR) methods are reviewed, forinstance, in “PCR protocols; A Guide to Methods and Applications”, Eds.Innis et al, 1990, Academic Press, New York, Mullis et al, Cold SpringHarbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR technology,Stockton Press, NY, 1989, and Ehrlich et al, Science, 252:1643-1650,(1991)). PCR comprises steps of denaturation of template nucleic acid(if double-stranded), annealing of primer to target, and polymerisation.

An amplification method may be a method other than PCR. Such methodsinclude strand displacement activation, the QB replicase system, therepair chain reaction, the ligase chain reaction, rolling circleamplification and ligation activated transcription. For convenience, andbecause it is generally preferred, the term PCR is used herein incontexts where other nucleic acid amplification techniques may beapplied by those skilled in the art. Unless the context requiresotherwise, reference to PCR should be taken to cover use of any suitablenucleic amplification reaction available in the art.

Sequencing

The polymorphism may be assessed or confirmed by nucleotide sequencingof a nucleic acid sample to determine the identity of a polymorphicallele. The identity may be determined by comparison of the nucleotidesequence obtained with a sequence shown in the Annex, Figures and Tablesherein. In this way, the allele of the polymorphism in the test samplemay be compared with the alleles which are shown to be associated withsusceptibility for osteoporosis.

Nucleotide sequence analysis may be performed on a genomic DNA sample,or amplified part thereof, or RNA sample as appropriate, using methodswhich are standard in the art.

Where an amplified part of the genomic DNA sample is used, the genomicDNA sample may be subjected to a PCR amplification reaction using a pairof suitable primers. In this way the region containing a particularpolymorphism or polymorphisms may be selectively amplified (PCR methodsand primers are discussed in more detail above). The nucleotide sequenceof the amplification product may then be determined by standardtechniques.

Other techniques which may be used are single base extension techniquesand pyrosequencing.

Mobility Based Methods

The assessment of the polymorphism may be performed by single strandconformation polymorphism analysis (SSCP). In this technique, PCRproducts from the region to be tested are heat denatured and rapidlycooled to avoid the reassociation of complementary strands. The singlestrands then form sequence dependent conformations that influence gelmobility. The different mobilities can then be analysed by gelelectrophoresis.

Assessment may be by heteroduplex analysis. In this analysis, the DNAsequence to be tested is amplified, denatured and renatured to itself orto known wild-type DNA. Heteroduplexes between different alleles containDNA “bubbles” at mismatched basepairs that can affect mobility through agel. Therefore, the mobility on a gel indicates the presence of sequencealterations.

Restriction Site Based Methods

Where an SNP creates or abolishes a restriction site, the assessment maybe made using RFLP analysis. In this analysis, the DNA is mixed with therelevant restriction enzyme (i.e., the enzyme whose restriction site iscreated or abolished). The resultant DNA is resolved by gelelectrophoresis to distinguish between DNA samples having therestriction site, which will be cut at that site, and DNA without thatrestriction site, which will not be cut.

Where the SNP does not create or abolish a restriction site the SNP maybe assessed in the following way. A mutant PCR primer may be designedwhich introduces a mutation into the amplification product, such that arestriction site is created when one of the polymorphic variants ispresent but not when another polymorphic variant is present. After PCRamplification using this primer (and another suitable primer), theamplification product is admixed with the relevant restriction enzymeand the resultant DNA analysed by gel electrophoresis to test fordigestion.

The invention will now be further described with reference to thefollowing non-limiting Figure, Example, Tables and Annex. Otherembodiments of the invention will occur to those skilled in the art inthe light of these.

FIGURES

FIG. 1 shows the TCIRG1 gene structure and location of polymorphisms.Common haplotypes with allele frequency greater than 5% are shown. Thetranslation start site (CDS) of the osteoclast specific isoform isindicated.

EXAMPLES OF BMD-RELATED TCIRG1 POLYMORPHISMS

Subjects

The study group comprised 739 unrelated women aged 45-55 who wererandomly selected from a large population based BMD screening programmefor osteoporotic fracture risk [15]. This screening program originallyinvolved 7000 women who were identified using Community Health Indexrecords (CHI) from a 25-mile radius of Aberdeen, a city with apopulation of about 250,000 in the North East of Scotland. Women wereinvited by letter to undergo BMD measurements between 1990-1994 and 5119of the 7000 invited (73.1%) attended for evaluation. Blood samples weresubsequently obtained for DNA extraction on 3069 (59.9%) of theseindividuals. Participants were weighed wearing light clothing and noshoes on a set of balance scales calibrated to 0.05 kg (Seca, Hamburg,Germany). Height was measured using a stadiometer (Holtain Ltd, Crymych,United Kingdom). Participants completed a questionnaire on menopausalstatus, and use of Hormone Replacement Therapy (HRT) and on the basis ofthis, were classified into five groups. Women were classified as“premenopausal” if they were not on HRT and menstruating regularly(n=374), as “perimenopausal” if they were not on HRT and menstruationwas irregular and/or if up to 6 months had elapsed since their lastperiod (n=14) and “postmenopausal” if they were not on HRT andmenstruation had ceased for 6 months or more (n=144). The remaining twogroups consisted of women who were currently receiving HRT at the timeof study (n=196) and those who previously had received HRT (n=11).Current and previous HRT users were not further classified in terms ofmenopausal status.

All participants gave written informed consent to being included in thestudy which was approved by the Grampian Joint Research EthicalCommittee.

Bone Mineral Densitometry

Bone mineral density measurements (BMD) of the left proximal femur (thefemoral neck, FN) and lumbar spine, LS (L2-4) were performed by dualenergy x-ray absorptiometry using one of two Norland XR26 or XR36densitometers (Norland Corp, Wisconsin, USA). Calibration of themachines was performed daily, and quality assurance checked by measuringthe manufacturer's lumbar spine phantom at daily intervals and a Hologicspine phantom at weekly intervals. The in-vivo precision for the XR36was 1.2% for the lumbar spine (LS), and 2.3% for the femoral neck (FN).Corresponding values for the XR26 were 1.95% and 2.31% (LS and FNrespectively).

Mutation Screening and Genotyping

Mutation screening was carried out by DNA sequencing of the promoter andintron-exon boundaries of the TCIRG1 gene in DNA extracted fromperipheral venous blood samples from about 70 individuals using PCRbased methods as previously described [13;14]. Genotyping forpolymorphisms was carried out by DNA sequencing of PCR amplifiedfragments of genomic DNA. The PCR products for sequencing were generatedusing Qiagen Taq DNA polymerase, Q-solution and standard reaction buffercontaining 1.5mM MgCl₂ according to the manufacturer's recommendations.The PCR was carried out for 35 cycles with a melting temperature of 95°C., an annealing temperature of 60° C. and an extension temperature of72° C.

The promoter polymorphisms (G9326A and G9508A); were analysed using thefollowing primer pairs: Forward: 5′ ACAAGGCAGGCGCAGGACTCC and Reverse:CGGGCCTGGAAACTGAGTCAC; the exon 4 (C14242T) and intron 4 (A14286G)polymorphisms were analysed using the following primer pairs: Forward 5′TTGGGGCAGCAGGTGGGGCC 3′ and Reverse: AGAGGAGAACCCCCTAGGGCTAG 3′; and theintron 11 polymorphism (G19031A) was analysed using the following primerpairs: Forward: GTTCGGGGATGTGGGCCAC 3′/and Reverse: 5′GCCCATAAGCAGGAGCAGG 3′. The PCR products were treated with ExonucleaseIII and Shrimp Alkaline Phosphatase-(Exo-SAP-IT) (Amersham Pharmacia)according to the manufacturers instructions and sequenced using theforward and/or reverse primer as the sequencing primer using DYNamic ETsequencing chemistry on a MegaBace 1000 DNA sequencer (AmershamPharmacia)

Statistical Methods

Statistical analysis was carried out using Minitab version 12 (MinitabInc, Pennsylvania, USA). Differences in BMD between the genotypes weretested using one way ANOVA and General Linear Model (GLM) analysis ofvariance (ANOVA) adjusting for height, weight, age, menopausalstatus/HRT use and smoking. Haplotypes were constructed from thepopulation genotype data by the algorithm of Niu and colleagues, usingthe Haplotyper program [Liu et al, (2002) Am.J Hum.Genet. 70:157-169].GLM ANOVA analysis was also used to test for allelic associations, bycombining data from the genotype groups and for haplotypes predicted bythe Haplotyper program. Stepwise logistic regression was used toevaluate the relative contribution of genotype and other factors to thepopulation variance in BMD. Linkage disequilibrium between polymorphismswas estimated by calculating D′ values using the 2BY2 program on outputgenerated by the EH program [Terwilliger J D, Ott J (1994) Handbook ofHuman Genetic Linkage. Johns Hopkins University Press, Baltimore &London]. Both programs were obtained from the Columbia UniversityWebsite.

Results

We identified 5 common polymorphisms (those with allele frequencygreater than 5%) in TCIRG1 on mutation screening of 70 normal subjects.These were: a C to T change at position 14242, (C14242T) an A to Gchange at position 14286 (Al4286G) and a G to A change at position 19031(G19031A) [which are positions 3856, 3900 and 8645 on sequence accessionnumber AF033033].

The C14242T change is within exon 4 of the osteoclast specifictranscript of the TCIRG1 gene but is a conservative change (CAC-CAT;both histidine). The A14286G polymorphisms is within intron 4 of TCIRG1and the G19031A is within intron 11 (G19031A). Two additionalpolymorphisms were discovered in the TCIRG1 promoter. These are atpositions 9326 (G9326A) and 9508 (G9508A) on sequence accession numberAP002807 in which the first nucleotide of the TCIRG1 mRNA start site isassumed to be position 10428. We did not detect any of the exonicpolymorphisms present in the SNP database cited by Carn et al[supra]with the exception of the C226T change which predicts an arginine totryptophan amino change at codon 56 (this is at position 2827 ofsequence accession number AF033033). This was rare however, with anallele frequency of only 2% in the normal subjects used for mutationscreening and was not analysed further in the population based study.

Details of age, BMD, height, weight, smoking history, menopausal status,and HRT use in the study population are shown in Table 1. 50.6% of thewomen were premenopausal, 1.8% perimenopausal and 19.4% werepostmenopausal. The average time elapsed since menopause was 6.07 yearsin the postmenopausal group. Menopausal status was unclassified for 207(28%) of subjects because the date of cessation of natural menstruationcould not be accurately established because of current HRT use in 196women (26.5%) and previous HRT use in 11 women (1.4%).

Significant linkage disequilibrium (LD) was observed between most of thepolymorphisms identified. The strongest LD was between G9326A and G9508A(D′=0.80, p<0.0001). Other LD values ranged between 0.569-0.752 (allp<0.001), with the exception of the C14242T polymorphism which showedsignificant LD only with A14286G. (D′=0.321; p<0.001). Analysis usingthe Haplotyper program predicted 27 different haplotypes from thegenotype data, but five common haplotypes were identified that accountedfor 77.3% of alleles at the TCIRG1 locus. These are summarised in FIG.1, which also illustrates the position of the polymorphisms in relationto the TCIRG1 gene structure.

We studied the relationship between genotypes at each site and BMDvalues, before and after adjustment for age, height, weight, menopausalstatus/HRT use and smoking. The results of this analysis are shown inTable 2 for spine BMD and Table 3 for hip BMD. The genotypedistributions of G9326A, G9508A, A14286G and G19031A were as predictedby Hardy-Weinberg equilibrium, but for the C14242T polymorphism, wefound more C/T heterozygotes than expected (102 vs 64, p=0.007).

There was a significant association between G9326A polymorphism and bothspine and hip BMD. The differences were significant for unadjusted andadjusted BMD values. When data were combining for the G/A heterozygotesand A/A homozygotes, the difference between groups was also significantat the spine and hip for adjusted BMD.

A non-significant trend for association between the C14242T polymorphismand adjusted spine BMD values was observed (p=0.079) and this becamesignificant when the C/T and T/T genotypes were combined (p=0.036). Noneof the other polymorphisms was associated with BMD, nor did we find asignificant association between any of the TCIRG1 haplotypes predictedby the Haplotyper program and BMD (data not shown). There was noassociation between TCIRG1 genotype and age, weight, height, smoking ormenopausal status (data not shown).

We also studied the relationship between TCIRG1 genotypes in relation tomenopausal status and HRT use. This analysis was restricted topremenopausal women, postmenopausal women and current HRT users in viewof the small number of subjects in the perimenopausal and previous HRTuser groups. There was no significant association between G9508A,C14242T, A14286G or G19031A genotypes and BMD in any of these subgroups,nor was there an association between TCIRG1 haplotypes and BMD (data notshown). The G9326A polymorphism was significantly associated with BMD inthe subgroup of women who were pre-menopausal, but there was noassociation between G9326A and BMD in postmenopausal women or HRT users(Table 4).

Analysis of the data by stepwise multiple regression identified threeindependent predictors of spine BMD, which together accounted for 13.3%of the variance in spine BMD. These were body weight (9.41% of thevariance, p<0.0001); menopausal status/HRT use (3.16% of the variance,p<0.0001); and the G9326A allele (1.00% of the variance, p=0.017). Forfemoral neck BMD, we identified two independent predictors whichaccounted for 11.1% of the variance. These were body weight (13.6% ofthe variance, p<0.0001) and menopausal status/HRT use (0.85% of thevariance, p=0.009). TABLE 1 Demographic details of study populationTable 1 Number 739 Age 47.9 ± 1.53 Premenopausal 374 (50.6%)Perimenopausal 14 (1.9%) Postmenopausal 144 (19.5%) Previous HRT users11 (1.5%) Current HRT users 196 (26.5%) Years since menopause* 6.1 ± 5.3Spine BMD (g/cm²) 1.067 ± 0.15  Femoral Neck BMD 0.890 ± 0.12  (g/cm²)Weight 65.9 ± 11.8 Height 161.5 ± 6.1  Values are means and SD or numbers and percentages.*in postmenopausal women

TABLE 2 Lumbar spine BMD values in relation to TCIRG1 genotypes andalleles ANOVA ANOVA Genotypes Allele (genotypes) (alleles) G9326A GG (n= 136) GA (n = 309) AA (n = 163) GA/AA p-value p-value (n = 472)(unadjusted BMD) 1.099 ± 0.173 1.052 ± 0.158 1.080 ± 0.140 1.062 ± 0.1520.011 0.017 (adjusted BMD) 1.074 ± 0.018 1.027 ± 0.016 1.049 ± 0.0181.035 ± 0.015 0.008 0.007 G9508A GG (n = 121) GA (n = 333) AA (n = 193)GA/AA (n = 526) (unadjusted BMD) 1.079 ± 0.181 1.059 ± 0.156 1.077 ±0.146 1.065 ± 0.153 0.311 0.379 (adjusted BMD) 1.061 ± 0.017 1.039 ±0.014 1.052 ± 0.016 1.043 ± 0.014 0.283 0.244 C14242T CC (n = 495) CT (n= 102) TT (n = 2) CT/TT (n = 104) (unadjusted BMD) 1.072 ± 0.157 1.043 ±0.148 1.070 ± 0.092 1.044 ± 0.147 0.240 0.094 (adjusted BMD) 1.049 ±0.014 1.014 ± 0.019 1.101 ± 0.103 1.015 ± 0.020 0.079 0.036 A14286G AA(n = 346) AG (n = 213) GG (n = 42) AG/GG (n = 255) (unadjusted BMD)1.095 ± 0.200 1.054 ± 0.151 1.074 ± 0.150 1.074 ± 0.150 0.187 0.326(adjusted BMD) 1.054 ± 0.015 1.031 ± 0.016 1.072 ± 0.025 1.039 ± 0.0150.109 0.200 G19031A GG (n = 428) GA (n = 160) AA (n = 21) GA/AA (n =181) (unadjusted BMD) 1.064 ± 0.153 1.066 ± 0.167 1.128 ± 0.161 1.073 ±0.167 0.189 0.530 (adjusted BMD) 1.038 ± 0.014 1.042 ± 0.016 1.102 ±0.034 1.048 ± 0.016 0.170 0.394

Unadjusted BMD values are mean±SD in g/cm². Adjusted BMD values areleast squares mean±SD BMD values adjusted for age, weight, height,menopausal status/HRT use and smoking. TABLE 3 Femoral neck BMD valuesin relation to TCIRG1 genotypes and alleles ANOVA ANOVA GenotypesAlleles (genotypes) (alleles) G9326A GG (n = 136) GA (n = 309) AA (n =164) GA/AA (n = 473) p-value p-value (unadjusted BMD) 0.907 ± 0.1440.879 ± 0.119 0.904 ± 0.123 0.888 ± 0.121 0.042 0.126 (adjusted BMD)0.896 ± 0.014 0.866 ± 0.013 0.886 ± 0.014 0.873 ± 0.012 0.030 0.047G9508A GG (n = 121) GA (n = 333) AA (n = 194) GA/AA (n = 527)(unadjusted BMD) 0.902 ± 0.147 0.880 ± 0.121 0.9508 ± 0.121  0.888 ±0.121 0.125 0.254 (adjusted BMD) 0.895 ± 0.014 0.871 ± 0.011 0.884 ±0.013 0.875 ± 0.011 0.114 0.104 C14242T CC (n = 496) CT (n = 102) TT (n= 2) CT/TT (n = 104) (unadjusted BMD) 0.894 ± 0.126 0.873 ± 0.117 0.865± 0.087 0.873 ± 0.117 0.301 0.122 (adjusted BMD) 0.874 ± 0.011 0.848 ±0.015 0.897 ± 0.082 0.849 ± 0.015 0.130 0.053 A14286G AA (n = 347) AG (n= 213) GG (n = 42) AG/GG (n = 255) (unadjusted BMD) 0.886 ± 0.123 0.888± 0.122 0.909 ± 0.145 0.892 ± 0.126 0.555 0.611 (adjusted BMD) 0.891 ±0.011 0.905 ± 0.013 0.932 ± 0.026 0.872 ± 0.012 0.474 0.787 G19031A GG(n = 429) GA (n = 160) AA (n = 21) GA/AA (n = 181) (unadjusted BMD)0.895 ± 0.121 0.876 ± 0.132 0.914 ± 0.130 0.881 ± 0.132 0.195 0.211(adjusted BMD) 0.878 ± 0.011 0.862 ± 0.012 0.9508 ± 0.027  0.865 ± 0.0120.179 0.234

Unadjusted BMD values are mean±SD in g/cm². Adjusted BMD values areleast squares mean±SD BMD values adjusted for age, weight, height,menopausal status/HRT use and smoking. TABLE 4 TCIRG1 G9326A alleles andBMD in relation to HRT use and menopausal status. G9326A G9326A ANOVAANOVA Genotypes Alleles (genotypes) (alleles) Spine BMD GG GA AA GA/AAp-value p-value Premenopausal (n = 308) (n = 67) (n = 157) (n = 83) (n =240) adjusted BMD 1.142 ± 0.020 1.075 ± 0.013 1.099 ± 0.019     1.082 ±0.012 0.011 0.006 Postmenopausal (n = 119) (n = 29) (n = 52) (n = 37) (n= 89) adjusted BMD 1.085 ± 0.026 1.065 ± 0.021 1.028 ± 0.024     1.049 ±0.016 0.230 0.228 HRT users (n = 164) (n = 32) (n = 94) (n = 37) (n =141) adjusted BMD 1.025 ± 0.025 1.016 ± 0.014 1.064 ± 0.024     1.028 ±0.013 0.215 0.863 Femoral Neck BMD GG GA AA GA/AA Premenopausal (n =308) (n = 67) (n = 157) (n = 84) (n = 240) adjusted BMD 0.934 ± 0.0150.882 ± 0.010 0.903 ± 0.014     0.888 ± 0.009 0.013 0.008 Postmenopausal(n = 119) (n = 29) (n = 52) (n = 37) (n = 89) adjusted BMD 0.896 ± 0.0200.904 ± 0.016 0.888 ± 0.018     0.897 ± 0.013 0.789 0.947 HRT users (n =164) (n = 32) (n = 94) (n = 37) (n = 141) adjusted BMD 0.869 ± 0.0210.869 ± 0.012  0.9508 ± 0.020    0.877 ± 0.011 0.379 0.708Adjusted BMD values are least squares mean±SD BMD values adjusted forage, weight, height, menopausal status/HRT use and smoking

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1. A method for assessing bone mass in an individual, the methodcomprising use of a T-cell immune regulator 1 (TCIRG1) marker.
 2. Amethod as claimed in claim 1 for assessing bone mineral density (BMD).3. A method as claimed in claim 2 wherein the method comprises(i)obtaining a sample of nucleic acid from an individual, and (ii)assessing a polymorphic marker in the TCIRG1 sequence of the nucleicacid, plus optionally one or more further steps to attribute a likelyBMD value to the individual.
 4. A method as claimed in claim 1 whereinthe marker is a polymorphic marker.
 5. A method as claimed in claim 3wherein the nucleic acid is genomic DNA.
 6. A method as claimed in claim5 wherein the marker is a single nucleotide polymorphism (SNP) selectedfrom the group consisting of the following positions numbered inaccordance with the ap002807 accession: (i) 9326; (ii) 9508; (iii)14242; (iv) 14286; (v) 19031, or a polymorphic marker which is inlinkage disequilibrium with any of these.
 7. A method as claimed inclaim 6 wherein the SNP is at position 9326, or is an SNP in linkagedisequilibrium with said SNP9326.
 8. A method as claimed in claim 7identity of the nucleotide at the SNP is assessed.
 9. A method asclaimed in claim 8 wherein the identity of the nucleotide at the SNP isone of the following alleles: (i) G9326A; (ii) G9508A; (iii) C14242T;(iv) A14286G; (v) G19031A.
 10. A method as claimed in claim 1 whereinthe individual is considered at risk from BMD-associated disorder.
 11. Amethod for determining the susceptibility of an individual to a disorderwhich is related to low BMD, the method comprising use of a method asclaimed in claim
 1. 12. A method as claimed in claim 11 wherein thedisorder is associated with a low lower lumbar spine or low femoral neckBMD.
 13. A method as claimed in claim 12 wherein the disorder isosteoporosis.
 14. A method as claimed in claim 13 wherein non TCIRG1polymorphic markers which are linked or associated with low BMD areassessed.
 15. A method for assessing the risk of osteoporotic fracturein an individual, the method comprising use of a method as claimed inclaim
 9. 16. A method as claimed in claim 15 wherein an individual whois A/A homozygous for SNP9326 or who is A/G heterozygous is classifiedas having increased risk and an individual who is G/G homozygous isclassified as having lowest risk, of susceptibility to a disorder whichis associated with an abnormally low BMD.
 17. A method as claimed inclaim 4 wherein the TCIRG1 sequence in assessed by determining thebinding of an oligonucleotide probe to the nucleic acid sample, whereinthe probe comprises all or part of (i) the TCIRG1 genomic sequence ofAnnex I, or (ii) a polymorphic form of the TCIRG1 genomic sequence shownin Annex I, or (iii) the complement of either.
 18. A method as claimedin claim 17 wherein the probe comprise a nucleic acid sequence whichbinds under stringent conditions specifically to one particular alleleof the TCIRG1 polymorphic marker and does not bind specifically toanother alleles of the TCIRG1 polymorphic marker.
 19. A method asclaimed in claim 18 wherein the probe is labelled and binding of theprobe is determined by presence of the label.
 20. A method as claimed inclaim 4 wherein the method comprises amplifying a region of the TCIRG1sequence comprising at least one polymorphic marker.
 21. A method asclaimed in claim 20 wherein a region of the TCIRG1 sequence is amplifiedby use of two oligonucleotide primers.
 22. A method as claimed in claim21 wherein at least one of said primers binds under stringent conditionsspecifically to one particular allele of the TCIRG1 polymorphic markerand does not bind specifically to another alleles of the TCIRG1polymorphic marker.
 23. A method as claimed in claim 21 wherein at leastone of said primers is a mutagenic primer which introduces a restrictionsite into said amplified region of the TCIRG1 sequence.
 24. A method asclaimed in claim 21 wherein at least one of said primers is selectedfrom: Forward: ACAAGGCAGGCGCAGGACTCC; (SEQ ID NO:2) Reverse:CGGGCCTGGAAACTGAGTCAC; (SEQ ID NO:3) Forward: TTGGGGCAGCAGGTGGGGCC; (SEQID NO:4) Reverse: AGAGGAGAACCCCCTAGGGCTAG; (SEQ ID NO:5) Forward:GTTCGGGGATGTGGGCCAC; (SEQ ID NO:6) Reverse: GCCCATAAGCAGGAGCAGG. (SEQ IDNO:7)


25. A method as claimed in claim 4 wherein the TCIRG1 sequence inassessed by a method selected from the group consisting of: strandconformation polymorphic marker analysis; heteroduplex analysis; RFLPanalysis.
 26. A method as claimed in claim 4 wherein the polymorphicmarker is assessed or confirmed by nucleotide sequencing,
 27. A methodof determining the presence or absence in a test sample of a polymorphicmarker in the TCIRG1 sequence which is a single nucleotide polymorphism(SNP) selected from the group consisting of the following positionsnumbered in accordance with the ap002807 accession: (i) 9326; (ii) 9508;(iii) 14242; (iv) 14286; (v) 19031, which method comprises determiningthe binding of an oligonucleotide probe to the nucleic acid sample,wherein the probe comprises all or part of (i) the TCIRG1 genomicsequence of Annex I, or; (ii) a polymorphic form of the TCIRG1 genomicsequence shown in Annex I, or; (iii) the complement of either.
 28. Amethod of determining the presence or absence in a test sample of apolymorphic marker in the TCIRG1 sequence which is a single nucleotidepolymorphism (SNP) selected from the group consisting of the followingpositions numbered in accordance with the ap002807 accession: (i) 9326;(ii) 9508; (iii) 14242; (iv) 14286; (v) 19031, which method comprisesuse of two oligonucleotide primers capable of amplifying a portion ofthe TCIRG1 sequence which portion comprises at least one of said SNPs.29. A method for mapping polymorphic markers which are associated with adisorder which is associated with an abnormal level of bone mineraldensity (BMD), the method comprising identifying polymorphic markerswhich are in linkage disequilibrium with an SNP which is selected fromthe group consisting of the following positions numbered in accordancewith the ap002807 accession: (i) 9326; (ii) 9508; (iii) 14242; (iv)14286; (v) 19031,
 30. An oligonucleotide probe for use in a method ofclaim
 21. 31. An oligonucleotide probe as claimed in claim 30 whichcomprises a TCIRG1 polymorphic marker which is a single nucleotidepolymorphism (SNP) selected from the group consisting of the followingpositions numbered in accordance with the ap002807 accession: (i) 9326;(ii) 9508; (iii) 14242; (iv) 14286; (v) 19031,
 32. An oligonucleotideprobe as claimed in claim 30 which comprises a label.
 33. A PCR primerpair for use in a method of claim 21 which primer pair comprises firstand second primers which hybridise to DNA in regions including orflanking the TCIRG1 polymorphic marker.
 34. A PCR primer pair as claimedin claim 33 wherein the TCIRG1 polymorphic marker is a single nucleotidepolymorphism (SNP) selected from the group consisting of the followingpositions numbered in accordance with the ap002807 accession: (i) 9326;(ii) 9508; (iii) 14242; (iv) 14286; (v) 19031,
 35. A PCR primer pair asclaimed in claim 34 wherein at least one primer is selected from:Forward: ACAAGGCAGGCGCAGGACTCC; (SEQ ID NO:2) Reverse:CGGGCCTGGAAACTGAGTCAC; (SEQ ID NO:3) Forward: TTGGGGCAGCAGGTGGGGCC; (SEQID NO:4) Reverse: AGAGGAGAACCCCCTAGGGCTAG; (SEQ ID NO:5) Forward:GTTCGGGGATGTGGGCCAC; (SEQ ID NO:6) Reverse: GCCCATAAGCAGGAGCAGG. (SEQ IDNO:7)


36. A kit comprising a probe andor primer of claim
 30. 37. A method ofosteoporosis prognosis, which method includes the step of screening anindividual for a genetic predisposition to osteoporosis in accordancewith the method of claim 13, whereby the predisposition is correlatedwith a TCIRG1 polymorphic marker, and if a predisposition is identified,treating that individual to prevent or reduce the onset of osteoporosis.38. A method as claimed in claim 37 wherein said treatment compriseshormone replacement therapy.